Project Summary/Abstract A major challenge in the field of tissue engineering is developing a functional vasculature to sustain the survival and engraftment of engineered tissues. Engineered human cardiac tissue has the potential to enhance heart function by replenishing cardiac muscle cells when used as a novel therapeutic for the treatment of heart disease, yet this new tissue requires a blood supply. Thus, our long-term goal is to induce an efficiently perfused, dense vascular bed in engineered human cardiac tissue to regenerate the myocardium. The overall objective of this proposal is to develop a method to encourage host endothelial cells to vascularize engineered tissues in vivo in pre-defined patterns. Our central hypothesis is that controlling the release kinetics and location of growth factors/cytokines will direct the migration of host endothelial cells to form a functional vasculature. We will test this hypothesis by performing a series of in vitro and in vivo analyses using an optimized, potent combination of growth factors and assessing endothelial cell morphogenesis and vessel formation. Aim 1 is to identify a set of growth factors/cytokines that maximize endothelial cell morphogenesis for vessel formation in vitro with supportive stromal/perivascular cells. Aim 2 is to control the spatiotemporal release of signaling molecules in engineered tissues to direct vascular development. Aim 3 is to evaluate perfusion through engineered tissue implants containing human induced pluripotent stem cell-derived cardiomyocytes and patterned growth factors/cytokines in vivo using 3D imaging to reconstruct the complete vascular supply to the heart and graft. Achieving these aims will improve our understanding of vascular development in response to growth factors/cytokines in the setting of cardiac regeneration and increase the perfusion and depth of penetration of vessels into engineered cardiac tissue. We believe this project is significant because it is expected to advance the development of a vascular supply to engineered myocardium for the purposes of heart regeneration. Further, this project is innovative in its utilization of patterned growth factors/cytokines in a controlled release biomaterial system in engineered cardiac tissue to facilitate vascular development in vivo.